Methods and apparatuses for structured illumination microscopy

Abstract

In structured illumination microscopy, the multiple recording of images with different phase positions of the structuring requires a high stability in the optical arrangement and sample throughout the entire measuring process. Also, the structuring must be projected into the sample in a highly homogeneous manner. The current invention optimizes recording of individual images in order to achieve the best possible resolution in the result image even in problematic samples. An optimization of this kind can be carried out in different ways, for example, by determining an optimal adjustment for at least one illumination parameter or recording parameter or by pulsed illumination such that an excitation from a triplet state of the fluorescent dye to a higher triplet state is reduced, or by illuminating the sample with depletion light for depopulating a triplet state of the fluorescent dye, which reduces bleaching.

Description

[0001]The present application claims priority from PCT Patent Application No. PCT / EP2009 / 006818 filed on Sep. 22, 2009, which claims priority from German Patent Application No. DE 10 2008 049 878.5 filed on Sep. 30, 2008, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The invention is directed to methods and devices for high-resolution microscopic imaging of a sample labeled with a fluorescent dye, wherein the sample is illuminated sequentially in a plurality of phases by structured, pulsed excitation light, and the fluorescent light emitted by the sample is recorded for each phase in a respective structured individual image so that a result image with enhanced resolution can be reconstructed from the individual images.[0004]2. Description of Related Art[0005]Due to the fact that the light received from the sample is diffracted in the microscope objective, the resolving power of microscopes ...

AI Technical Summary

Benefits of technology

[0047]In particularly preferred embodiment forms of the methods according to the invention, the sample is excited by structured line illumination. This makes it possible to use high excitation powers.

[0048]In so doing, an intensity of the line illumination is advantageously adjusted in such a way that a dynamic range of a recording camera is fully utilized and the intensity is stored line by line. A high dynamic range means a high signal-to-noise ratio and, therefore, a high resolution.

[0049]The fluorescent light is preferably detected confocally, particularly by means of a laser scanning microscope. In this way, out-of-focus light is efficiently discriminated and the signal-to-noise ratio is accordingly improved. By combining confocal detection with structured line illumination, background which may possibly be disruptive for the feedback is reduced already during image recording.

Problems solved by technology

Since the usable wavelength range of visible light is finite, the resolving power of a microscope is fundamentally limited (Abbe, 1873).

Further, the required multiple recording reduces the effective frame rate.

As a result, distinctly different nonlinearities may be achieved at different locations in the sample, which makes reconstruction of the result image more difficult.

In some cases, the environmental conditions in the sample can be so unfavorable for a nonlinear interaction that the use of SPEM is impossible.

In SIM and SPEM, the resolution which can be achieved by reconstruction is fundamentally limited by the signal-to-noise ratio (SNR) in the recording of individual images.

Images